Lysozyme (EC 3.2.1.17), also named muramidase, is a hydrolase acting on microbial cell walls, and is able to effectively hydrolyze the peptidoglycan of the bacterial cell wall by cleaving the β-1,4-glycosidic bond between the N-acetylmuramic acid and the N-acetylglucosamine of the peptidoglycan. Consequently, the cell wall is degraded due to the breakage of the peptidoglycan backbone, which results in lysis of the bacterial cell. The lysozyme is widely distributed in nature, such as in tears, saliva, snot and tissues of mammalians and in egg white of birds and poultries. Based on different resources, the lysozymes are divided into six types: chicken-type (c-type) lysozyme, goose-type (g-type) lysozyme, invertebrate-type (i-type) lysozyme, phage-type lysozyme, plant lysozyme and bacterial lysozyme. The lysozyme is a non-toxic protein and has no side effects to humans and mammalians. Due to the lytic property, the lysozyme is widely used in different industries these years. In dairy industry, the lysozyme can be used as a nature preservative; for example, adding lysozyme into the pasteurized milk can extend its shelf life. In food industry, adding lysozyme can extend the storage time of aquatic products and meat foods. In animal feed industry, adding lysozyme can improve animal production performance. According to studies, adding lysozyme into piglet diets can increase animal feed conversion, reduce diarrhea rates, improve piglet's health and reduce the use of antibiotics.
In recent years, due to global abuses of antibiotic drugs, more and more bacteria have developed into drug-resistant strains. Therefore, many countries start to prohibit the addition of antibiotics to livestock and poultry feeds, and scientists are trying hard to find solutions to replace traditional antibiotics, which draws more attention to lysozyme research. So far, many researches attempt to obtain lysozyme that can satisfy various industrial applications by either screening new genes from nature or modifying existing enzymes. In many strategies for modifying the enzyme, the rationale engineering that specifically mutates the enzyme gene based on the structural information of the enzyme protein is one of the major methods of modifying the enzyme. In this strategy, to improve the enzymatic activity is also a major focus of the industrial enzyme improvement. The improvement of the enzyme activity represents the reduction of the cost and the increase of the profit, and the enzyme having improved enzymatic activity is also beneficial to industrial applications.
Therefore, the present invention intends to improve the enzymatic activity of the lysozyme by rationale engineering, so as to further increase the industrial value of the lysozyme.